Discovery of single-nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete Ophiognomonia clavigignenti-juglandacearum from 454 sequence data

The benefits from recent improvement in sequencing technologies, such as the Roche GS FLX (454) pyrosequencing, may be even more valuable in non-model organisms, such as many plant pathogenic fungi of economic importance. One application of this new sequencing technology is the rapid generation of genomic information to identify putative single-nucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. The focus of this research was to sequence, assemble, discover and validate SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. Genomic DNA from eight isolates of Ophiognomonia clavigignenti-juglandacearum was pooled in one region of a four-region sequencing run on a Roche 454 GS FLX. This yielded 71 million total bases comprising 217,000 reads, 80% of which collapsed into 16,125,754 bases in 30,339 contigs upon assembly. By aligning reads from multiple isolates, we detected 298 SNPs using Roche's GS Mapper. With no reference sequence available, however, it was difficult to distinguish true polymorphisms from sequencing error. Eagleview software was used to manually examine each contig that contained one or more putative SNPs, enabling us to discard all but 45 of the original 298 putative SNPs. Of those 45 SNPs, 13 were validated using standard Sanger sequencing. This research provides a valuable genetic resource for research into the genus Ophiognomonia, demonstrates a framework for the rapid and cost-effective discovery of SNP markers in non-model organisms and should prove especially useful in the case of asexual or clonal fungi with limited genetic variability.     

‘Escaping' the X chromosome leads to increased gene expression in the male germline of Drosophila melanogaster

Genomic analyses of Drosophila species suggest that the X chromosome presents an unfavourable environment for the expression of genes in the male germline. A previous study in D. melanogaster used a reporter gene driven by a testis-specific promoter to show that expression was greatly reduced when the gene was inserted onto the X chromosome as compared with the autosomes. However, a limitation of this study was that only the expression regulated by a single, autosomal-derived promoter was investigated. To test for an increase in expression associated with ‘escaping'' the X chromosome, we analysed reporter gene expression driven by the promoters of three X-linked, testis-expressed genes (CG10920, CG12681 and CG1314) that were inserted randomly throughout the D. melanogaster genome. In all cases, insertions on the autosomes showed significantly higher expression than those on the X chromosome. Thus, even genes whose regulation has adapted to the X-chromosomal environment show increased male germline expression when relocated to an autosome. Our results provide direct experimental evidence for the suppression of X-linked gene expression in the Drosophila male germline that is independent of gene dose.